This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ULTRASTRUCTURAL BASIS FOR SYNAPTIC VESICLE RECYCLING IN THE CALYX OF HELD The calyx of Held and other large nerve terminals of the auditory brainstem are key elements of sound localization circuitry. Our goal is to reveal structural transformations and cellular communication that characterize contact of the calycigenic growth cone with its target and the early stages of synapse assembly and stabilization at large nerve terminals in the auditory brainstem. Our central hypothesis is that competition among calycigenic inputs precedes expansion of the terminal over the cell body to form a calyx. This hypothesis is based on work from our laboratory that reveals rapid formation of the calyx in mice between postnatal days (P)2 and P4. The calyx contains hundreds to greater than two thousand active zones, depending upon the species, many of which are located nearby specialized organelle complexes termed mitochondrion-associated adherens complexes (MACs). MAC structure had been described previously using standard transmission electron microscopy (sTEM), which revealed filaments tethering the mitochondrion to a punctum adherens that links the pre- and postsynaptic membranes. Confocal fluorescence imaging and electron tomography are being employed to study medial nucleus of the trapezoid body (MNTB) cell innervation during P0[unreadable]P4. This time period precedes and overlaps the formation of immature, cup-shaped calyces that envelop the MNTB cell body. These experiments will pinpoint time periods during which mono-innervation is established between pre- and postsynaptic partners and highlight structural and functional differences that may predict winning and losing inputs. In addition, we will describe structural features of mature calyces that support high-rate neurotransmission that is characteristic of this terminal. Because of the size of the calyx-MNTB contact and the desired high resolution of the reconstructions, serial volumetric imaging of domains of cells will be required. This project will require high-resolution, wide-field, large-area digital recording of images for electron tomography.